The identification of potent macromolecules is increasing at a staggering rate, however many are not delivered effectively due to physiochemical limitations. A viable approach to drug delivery may be to exploit the physiological regulation of intercellular tight junctions, to enhance paracellular drug transport. We have recently demonstrated that Zonula Occludens Toxin (Zot) Zot, a 45kDa protein, exerts its permeating effect by mimicking a eukaryotic analogue in charge of modulation of intercellular tight junctions This technology allows for enhanced paracellular flux and has the potential to effectively deliver low bioavailable therapeutic macromolecules. Thus our hypothesis is that the Zot technology can enhance drug delivery by reversibly opening tight cellular junctions via oral, BBB and nasal delivery. The following specific aims will be pursued. SAI. To define the structure-function relationships between the major domains of Zot and regulation of the transepithelial/endothelial paracellular pathway. SA2. To determine the dose response, pharmacokinetics (PK) and acute toxicity of Zot. We will define the complete dose-response profile of the Zot (or derivative) in vivo. SA3. To examine the ability of Zot to enhance the oral bioavailability of the hydrophilic agents (mannitol, inulin) and therapeutic macromolecules (insulin, cyclosporin A, paclitaxel). Preliminary data displays that Zot enhances oral absorption of therapeutic molecules. SA4. To examine the ability of Zot to enhance the BBB delivery of the minimally transported CNS therapeutic macromolecules. Zonulin, the ligand for the Zot receptor has been found in the brain and data has shown that Zot enhances the BBB delivery of therapeutic agents (doxorubicin, inulin, acyclovir). SA5. To examine the ability of Zot to act as a novel mucosal adjuvant that enhances the systemic bioavailability of large molecular weight therapeutic macromolecules (e.g., peptides, Protective antigen to B. anthracis) after nasal delivery. Zot has been found to significantly enhance the production of tetanus toxin (TT) antibodies after nasal delivery. The in vivo studies proposed for SA 3-5 will evaluate the ability of Zot to enhance the systemic levels of structurally diverse therapeutic macromolecules. The potential impact of the Zot technology is significant and comprehensive, since it may be applied to delivering a diversified range of macromolecules to their targets via oral, BBB or nasal delivery.